The objective of the research proposed is to further our understanding of the mechanisms used to repress transcription in eukaryotic cells. Regulatory transcription factors (TF) generally require two separate domains or motifs to act as repressers: a DNA binding domain (DBD) and a repression domain (RD). The DBD allows the factor to bind in a sequence specific manner to elements adjacent specific target genes, while RDs employ a variety of mechanisms to silence transcription of the adjacent gene. Many, if not most, RDs act indirectly by recruiting accessory proteins known as corepressors (CoRs) which possess the activity necessary to repress transcription;in particular they may have the ability, or recruit other proteins that have the ability, to covalently modify histone tails leading to a compaction of chromatin structure or an alteration in the binding of chromatin associated proteins involved in transcriptional control. Two main questions will be addressed, one general and one more specific. In theory, a single RD could provide all the activity required for a TF to function, but real TFs such as the Brinker (Brk), Engrailed (En) and C15 proteins of Drosophila possess multiple RDs and appear to be able to recruit more than one corepressor. Consequently, the general question to be addressed is why do most TFs possess more than one RD, allowing them to recruit more than one co-repressor? The second, more specific question is how do different corerpessors more precisely, Groucho (Gro), C-terminal Binding Protein (CtBP), Atrophin (Atro), and Scribbler (Sbb), actually function to repress transcription? There are two Specific Aims: (1) To determine why Brk requires multiple repression domains and characterize CtBP and Gro- dependent repression by Brk. We will investigate in detail why one transcription factor, Brk, possesses more than one repression domain by determining the function of each in vivo. We will investigate how CtBP and Gro provide activity to Brk in particular how they act over distance. (2) To define the roles of Atro and Sbb in transcriptional repression. Although Atro is an important CoR in both flies and vertebrates, we know little about how it functions. We will investigate basic properties of Atro- dependent repression and identify factors required to achieve this, in particular the role played by Sbb. Understanding the mechanisms of transcriptional repression has important implications for human health as defects in this process can lead to human disease, in particular cancer and neurodegenerative disorders. For example, expansion of a polyglutamine tract in the human homolog of Atro is the cause of an inherited neuronal degenerative disease, dentatorubral-pallidoluysian atrophy, while misexpression of Hox11, the human homolog of C15, results in specific T-cell leukemias in humans. Consequently, the proposed studies should provide insights into diseases such as these, potentially leading to improved diagnosis and treatment.